Studies indicate that significant inter-individual variation exists at the amino acid sequence level of BER proteins, and that variability exists in the BER capacity within the population. We have initiated efforts to delineate the impact of amino acid variants found in BER proteins and to develop assays to determine the extent of inter-individual variation in specific steps of BER. For example, we are currently investigating whether variants found in the major human abasic endonuclease (APE1), either within the normal population or that are tumor-associated, affect protein function and may therefore contribute to disease formation and/or progression. Our most recent work has revealed that except for the endometrial cancer-associated APE1 variant R237C, the polymorphic variants Q51H, I64V and D148E, the rare population variants G241R, P311S and A317V, and the tumor-associated variant P112L exhibit normal thermodynamic stability of protein folding; abasic endonuclease, 3'-5' exonuclease and REF-1 activities; coordination during the early steps of base excision repair; and intracellular distribution when expressed exogenously in HeLa cells. The R237C mutant displayed reduced AP-DNA complex stability, 3'-5' exonuclease activity and 3'-damage processing. Re-sequencing of the exonic regions of APE1 uncovered no novel amino acid substitutions in the 60 cancer cell lines of the NCI-60 panel, or in HeLa or T98G cancer cell lines; only the common D148E and Q51H variants were observed. Our results indicate that APE1 missense mutations are seemingly rare and that the cancer-associated R237C variant may represent a reduced-function susceptibility allele. Using established biochemical assays, we have also begun to evaluate whether inter-individual variation in BER associates with disease susceptibility and/or clinical agent responsiveness; we are also assessing for age-dependent or gender-specific variation. Current biochemical methods have allowed us to interrogate the central steps of BER. Our results indicate that for AP site incision, the twenty-three individuals examined thus far exhibit an 1.9-fold inter-individual variation in repair capacity. For gap-filling and nick ligation, we observed an 1.3-fold and 3.4-fold inter-individual variation, respectively. The greater disparity in nick ligation stems at least in part from the low overall activity and the corresponding limited sensitivity of the assay. Like the AP endonuclease profile, the average repair capacity of each individual for gap-filling and nick sealing generally fell within the experimental variability of the population, suggesting a comparatively similar BER efficiency among this small group of samples. We are presently designing a high throughput BER pathway assay to more robustly evaluate the relationship of repair capacity with disease susceptibility and premature aging phenotypes. Current strategies to eradicate cancer cells typically employ agents that generate DNA lesions that induce cell death by blocking replication of rapidly dividing cells. Thus, a goal has been to regulate strategically the repair capacity of cancer and/or normal cells to improve the efficacy of specific therapeutic paradigms. In particular, inhibiting the DNA repair capacity of cancerous cells has been an area of promising interest. Our recent results indicate that APE1, and BER more generally, is a reasonable target for inactivation in anti-cancer treatment paradigms involving select alkylating drugs (e.g., temozolomide) and antimetabolites (e.g., 5-fluorouracil). We have recently described the development of a panel of complementary and improved miniaturized high-throughput screening and profiling assays, which will have a broad appeal to other research investigators, particularly those in the field of DNA repair. These assays have permitted the identification of novel, small molecule APE1-targeted bioactive inhibitors, which we are working to optimize with the long term goal of creating high affinity, selective inhibitors with therapeutic value. The establishment of small molecule probes will provide a platform for more extensive investigations on the therapeutic benefits of regulating cellular DNA repair capacity. Although the work so far has focused on the design of DNA repair inhibitors, future studies are aimed at the development of DNA repair pathway activators. While we are at the very early stages of conceptualizing appropriate methods, one could, for example, screen for compounds that increase resistance to a relevant DNA-damaging agent, such as an oxidizing agent. Another idea would be to identify small molecules that reduce the levels of a specific DNA damage marker (e.g., strand breaks or abasic sites) after exposure to a particular DNA-damaging agent. In addition to compounds that activate genotoxic responses, one could envision screening for molecules that improve mitochondrial function or reduce oxidative stress, end-points relevant to aging. Stimulation of genome maintenance mechanisms, or more broadly, effective stress responses, has the potential to provide healthspan benefits.